Project Summary Development of blood cell lineages from hematopoietic stem cells (HSC) is stringently regulated, and defects in this process cause diseases including blood cancers like acute myelogenous leukemia (AML), the most lethal leukemia subtype. Faced with dynamic and potentially oncogenic perturbations in their bone marrow microenvironment, how HSC progenitors balance self-renewal with differentiation and preserve their integrity remains a central question in hematopoiesis. The highly conserved endoplasmic reticulum-membrane kinase- endoribonuclease IRE1? (inositol-requiring enzyme-1), acting through the product of its mRNA substrate X-box binding protein (Xbp)-1, orchestrates an ?unfolded protein response? program that facilitates ER protein folding in eukaryotic cells. Beyond this role in cellular proteostasis, I recently discovered that the IRE1?/Xbp1 pathway is a critical cell-intrinsic brake against myeloid leukemogenesis in HSCs. In a mouse model of the Flt3 internal tandem duplication of the juxtamembrane region (Flt3-ITD), the most frequent mutation in AML, HSC- specific loss of IRE1?/Xbp1 caused a lethal AML not seen in WT Flt3-ITD mice. Consistent with these findings, we found that downregulation of an IRE1?/Xbp1 target gene signature was associated with poor overall survival in a cohort of AML patients, revealing the novel possibility that dysregulated IRE1?/Xbp1 signaling importantly contributes to AML pathogenesis. Preliminary transcriptome analysis of IRE1?-deficient HSCs revealed a marked upregulation of Wnt/?-catenin signaling, a key pathway required for the development and function of the ?so-called? leukemia stem cells (LSCs), few quiescent and self-renewing cells that initiate, maintain and are implicated in AML relapse but are rarely targeted by current therapies. As IRE1? is a convergence point and sensor for various cellular perturbations (e.g. reactive oxygen species, hypoxia, nutrient deprivation, chemicals, cytokines and metabolites), our findings suggest a novel paradigm in which activation of IRE1? in response to these perturbations restrains the self-renewal potential of HSC progenitors to mitigate leukemogenesis. Using mouse AML models integrating IRE1? activity reporter and inducible Xbp1 transgene that I recently developed, we will address how the IRE1?/Xbp1 pathway is regulated during hematopoiesis (Aim1) and the molecular mechanisms by which this pathway suppresses myeloid leukemogenesis (Aim 2). There is increasing appreciation that because of their important role in AML pathogenesis, eradication of LSCs will be critical to achieve long-term AML remission. As IRE1?/Xbp1 suppresses the Wnt/?-catenin signaling required for LSC function, we will test the hypothesis that targeted activation of the IRE1?/Xbp1 pathway in HSC progenitors will limit LSC development and function (Aim 3), providing an innovative strategy for durable AML therapy. These studies are integral to my long-term commitment to run a strong successful independent research laboratory focused on understanding normal and malignant blood cell development with the ultimate goal of discovering improved therapies for blood cancers.